L-arginine is a semi-essential amino acid in human and animal bodies, is a synthesis precursor of various bioactive substances, and has various unique physiological and pharmacologic effects. With the continuous deep research and understanding of the biological function of arginine, arginine is more and more widely applied to medicines, food and feed industry.
Production methods of L-arginine include a hydrolysis method and a fermentation method. Currently, a protein hydrolysis method is still mainly adopted by most of existing manufacturers to produce L-arginine in China, and the method is serious in environment pollution and not high in yield, so as not to be suitable for large-scale production. The fermentation method for producing L-arginine is relatively simple in process and environment-friendly, so as to have a great development potential. However, a method for producing L-arginine by microbial fermentation domestically is generally relatively low in acid production level and relatively high in cost, and the production level and the yield cannot meet domestic demands. Therefore, it is very important to improve the fermentation acid production level of L-arginine and increase the utilization ratio of glucose.
Corynebacterium crenatum SDNN403 (the strain with a collection number of CGMCC NO:0890 and a patent number of ZL 03112896.3 is researched for many years by the research group, and is obtained by using a traditional mutagenesis method) is high-yield L-arginine C. crenatum. The anabolic pathway of L-arginine of the strain is systematically analyzed in preliminary work. The effect on regulating feedback inhibition and feedback repression in anabolism of L-arginine is researched to relieve feedback inhibition in the strain. Meanwhile, key metabolizing enzyme genes on a competition bypath for synthesizing arginine are subjected to metabolic transformation such as knockout, so that metabolic flows on competition branch pathways of proline, glutamine and the like are weakened while the expression of a key enzyme gene cluster on a synthesis pathway of arginine serving as a target product is enhanced, finally, the catabiosis of L-arginine is concentrated on an anabolic flow of L-arginine, and furthermore, the yield of arginine is increased.
Active oxygen comprises superoxide anions O2−, hydroxyl radicals OH., hydrogen peroxide H2O2 and the like which are by-products in a biological aerobic metabolism process, and have very high toxicity for cells. After evolving for a long term, the cells have formed a set of action system for resisting the toxicity of active oxygen, for example, superoxide dismutase can be used for converting the superoxide anions O2− into H2O2, and catalase can be used for decomposing H2O2 into H2O and O2. It is a passive defense process that the cells are decomposed after generating active oxygen, it is impossible to immediately eliminate produced active oxygen, and a small amount of active oxygen can still react with intracellular substances to damage the cells. If the synthesis of active oxygen can be directly reduced, the damage of active oxygen to the cells can be fundamentally reduced.
As one of active oxygen in the cells, H2O2 has very high oxidation activity and toxicity. It is reported that flavin reductases can be used for reducing oxidized flavins FMN and FAD as well as riboflavin into reduced flavins FMNH2 and FADH2 as well as reduced riboflavin by utilizing NAD(P)H, and then further carrying out catalysis to transfer electrons of reduced flavins to other intracellular electron acceptors. If the electron acceptors are O2, it is possible to produce H2O2.
Found by retrieving a genome of Corynebacterium glutamicum ATCC13032, hypothetic NADPH-dependent FMN reductase is encoded by a gene cg1150 (genome NCBI Accession Number: BX927151) and a gene cg3223 (genome NCBI Accession Number: BX927156). However, functions of the two genes are not clear so far, and no correlated researches are reported.
Although it has been reported currently that the metabolic pathway of the strain is transformed to increase the yield of L-arginine, it is necessary to develop a novel method to further promote the synthesis of L-arginine.